(341d) Developing a Stochastic Model of LPS-Induced TNF-α Production in Macrophages

Macrophages are immune cells that are present throughout the body and are involved in a variety of physiological responses. One of its most important roles is its regulation of innate and adaptive immune responses [1]. Toll-like receptors (TLR) embedded in the cell membrane mediate these responses by recognizing microbial components in the body. One such microbial component is LPS (lipopolysaccharide), which is a major constituent of bacterial membranes [2]. Binding of LPS to TLRs leads to the initiation of intracellular signaling pathways and downstream gene expression to rapidly eliminate the bacterial components. One of the major signaling pathways activated by TLR signaling is through the transcription factor NF-κB, which leads to the production of the pro-inflammatory cytokine TNF-α (tumor necrosis factor-α). TNF-α, in turn, initiates signaling through the TNF signaling pathway and leads to propagation of the inflammatory response to microbial components. Hence, LPS-induced signaling pathway is an important part of the early immune response.

Previous studies [3, 4, 5] have formulated population-level models to study the dynamics of signaling through the NF-κB pathway. In these models, the numerical model predicts population-level activation of NF-κB and downstream TNF-α gene expression. However, predictions from the population-level model could be misleading since the model masks the behaviors of individual cells. It has been shown that the effect of LPS stimulation can be highly stochastic, especially to low concentrations of LPS where only a fraction of cells respond to stimulation [6]. Therefore, we developed a stochastic model of LPS-induced NF-κB signaling and TNF-α production in macrophages. Specifically, in addition to gene activation and cellular membrane receptor activation, enzymes and proteins involved in the intermediate stages of LPS-induced NF-κB signaling were modeled as states. Then, the corresponding kinetic parameters for intracellular signaling pathway were identified via the sequential approach of model reduction and inverse modeling. Model simulations were validated and refined using experimental data on LPS stimulated macrophages. Specifically, the activation of NF-κB, TNF-α and IκB in RAW macrophages upon stimulation with different LPS doses was studied using flow cytometry and intracellular staining at the single-cell level. These models are expected to predict the diversity of cellular responses that cannot be captured by deterministic and population-based models. Furthermore, it was possible to simulate intercellular interactions with this stochastic model to analyze cell-to-cell communications - a step toward constructing the multicellular or tissue model. More importantly, the developed stochastic model was used to design a feedback control system that helps producing TNF-α at the desired rate through the autocrine regulation of the LPS-induced intracellular signaling pathway in macrophages.

References:

[1] Parameswaran N, Patial S. 2010. Tumor Necrosis Factor-α Signaling in Macrophages. Crit Rev Eukaryot Gene Expr. 20: 87-103.

[2] Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K, Akira S. 1999. Differential Roles of TLR2 and TLR4 in Recognition of Gram-Negative and Gram-Positive Bacterial Cell Wall Components. Immunity 11: 443-451.

[3] Choudhary S, Kalita M, Fang L, Patel K, Tian B, Zhao Y, Edeh C.B, Brasier A.R. 2013. Inducible TNF receptor associated factor-1 expression couples the canonical to non-canonical NF-κB pathway in TNF stimulation. Journal of Biological Chemistry 288:14612-14623.

[4] Lipniacki T, Paszek P, Brasier A.R, Luxon B, Kimmel M. 2004. Mathematical model of NF-κB regulatory module. Journal of Theoretical Biology 228: 195-215.

[5] Shih V, Kearns D, Basak S, Savinova O, Ghosh G, Hoffmann A. 2009. Kinetic control of negative feedback regulators of NF-κB/RelA determines their pathogen- and cytokine-receptor signaling specificity. Proceedings of the National Academy of Sciences 106: 9619-9624.

[6] James C, Moorman M, Carson B, Branda C, Lantz J, Manginell R, Martino A, Singh A. 2009. Nuclear translocation kinetics of NF-κB in macrophages challenged with pathogens in a microfluidic platform. Biomed Microdevices 11: 693-700.